Oxidative damage to DNA is caused spontaneously by normal by-products of metabolism and is induced by exposure to ionizing radiation and environmental agents. Reduced ability to prevent or repair oxidative DNA damage has been implicated in a number of human diseases, including cancer and neurodenerative diseases, and contributes to the deleterious consequences of aging. We have developed a simple screen that allows the identification of human genes that prevent or repair DNA oxidative damage. This screen detects human genes by their ability to complement the spontaneous mutator or phenotype when expressed in bacteria deficient in repair of oxidative damage. It will be used to attain the long term goals of developing a thorough understanding of oxidation protection mechanisms by identifying and characterizing human oxidative DNA damage prevention and repair genes and determining their mode of action. Using this screen in a preliminary search, we have identified two new human genes, OXR1 and PC4, that can prevent oxidative mutagenesis in bacteria. An additional gene, OXR2, has been identified using a genomic approach to search the human genome for additional OXR1 related genes. It has extensive homology to OXR1 and contains the highly conserved OXR domain. We have demonstrated that OXR2 can also function in oxidation protection. By producing knockout mutations of the yeast homologues of OXR1 and PC4, we demonstrated that these genes are required in eukaryotes for resistance to treatments with the oxidative agent hydrogen peroxide. In yeast and mammalian cells, OXR1 is a peroxide and heat stress responsive protein that localizes to mitochondria, whereas OXR2 is its constitutive, nuclear counterpart. Preliminary data suggests PC4functions in an XPG dependent DNA repair pathway acting on oxidative DNA damage. We will now determine how these genes function to provide resistance to oxidative DNA damage in yeast and mammalian cells by determining their effects on the oxidative state of the cell, damage production by peroxide, and by examining their possible roles in specific DNA repair pathways. We will also characterize their activities in a variety of oxidation related cellular processes, including effects on spontaneous and peroxide induced mutagenesis, heat shock tolerance and mutagenesis, and peroxide induced apoptosis.